The goal of this project is to design and express novel human mutant recombinant hemoglobins (rHbs), which, after reacting with polyethylene glycol (PEG) or other cross-linking reagents, possess appropriate structural and functional properties to serve as hemoglobin-based oxygen carries (HBOCs). The vasoactivity associated with the use of many of the current HBOCs has challenged previous beliefs as to the optimal O2 affinity, cooperatively, Bohr effect, size, viscosity, and other properties needed for HBOCs. rHbs that we have previously prepared and new ones that we will design and express will be used to test both the new paradigms for the design of blood substitutes and the various hypotheses regarding the origin of vasoactivity. Using our Hb expression system in Escherichia coli, we are in a position to design and express any rHbs needed. Our laboratory has extensive experience and is also fully equipped to carry out structural studies using nuclear magnetic resonance (NMR) and functional studies using biochemical-biophysical techniques (e.g., equilibrium oxygen binding, kinetics of ligand binding, etc)on Hbs. A unique feature of this Program Project is the interactions among its component parts. The key investigators of this Program Project have worked together and have published papers together during the past ten years. The scope of this Program Project requires a multidisciplinary approach to the design of novel HBOCs. The structural and biochemical-biophysical results obtained from this project (Project 2) will provide new insights for Projects 1, 3, 4 and 5 as well as the Protein Biochemistry Core. Equally, the results from other components of the Program Project will guide Project 2 in designing the rHbs most effective as HBOCs. We believe that the results obtained from this Program Project will provide new knowledge for the development of safe, reliable HBOCs as blood substitutes.